Over the past fiscal year, we have performed studies on three viral pathogens causing respiratory disease in humans, Nipah virus, influenza virus and Middle East Respiratory Syndrome coronavirus (MERS-CoV). (1) To develop animal disease (end host) and persistence (reservoir host) models: We have developed a nonhuman primate model for the Bangladesh genotype of Nipah virus in African green monkeys. The availability of this model is of great importance since most human cases of Nipah virus since 1999 were caused by viruses of the Bangladesh genotype. The results suggest that the pathogenesis of this genotype is like that of the Malaysia genotype, but with accelerated disease progression in the end stage. This model will be instrumental for ongoing and future studies on vaccines and antivirals (Lo et al., Sci Transl Med 2019; de Wit et al., unpublished) (2) To identify and characterize determinants of viral pathogenicity to develop antivirals: In collaboration with the Molecular Targets Program at NCI, griffithsin, a novel viral entry inhibitor, was identified as having potent (EC50 5nM) activity against MERS-CoV. The post-exposure efficacy of nebulized griffithsin in the rhesus macaque model showed moderate reduction of viral load but did not significantly reduce disease signs. We have now shown that pre-exposure treatment reduces clinical signs of disease and viral titers in target organs. (Falzarano et al., manuscript in preparation) We have tested the efficacy of the antiviral compound GS-5734 against MERS-CoV in the rhesus macaque model. Pre-exposure treatment resulted in reduction of disease burden and viral lung loads. In contrast, post-exposure treatment with GS-5734 showed only minor effects. Thus, the compound can be considered for prophylactic applications. (de Wit et al., manuscript submitted) We also tested the efficacy of GS-5734 against Nipah virus (Bangladesh genotype) in African green monkeys. Animals were inoculated with a lethal dose of Nipah virus and a once-daily intravenous GS-5734 treatment was initiated one day later. Mild respiratory signs were observed in 2 of 4 treated animals, whereas all control animals developed severe respiratory disease signs. In contrast to control animals, which all succumbed to the infection, all GS-5734-treated animals survived the lethal challenge, indicating that GS-5734 is a promising antiviral treatment for Nipah virus infection. (Lo et al. Sci Transl Med 2019) The observed virulence of the 1918 influenza virus substantiates the need for evaluating antiviral options against infections with this virulent human pathogen. Here we demonstrate that oseltamivir phosphate, which has never been evaluated in macaques against infection with the fully reconstructed 1918 influenza virus, is effective in preventing severe disease in cynomolgus macaques if given prophylactically. Efficacy was reduced in a treatment regimen through emergence of oseltamivir-resistant mutants that lead to death of one of four animals. This emphasizes the importance of implementing combination therapy and vaccination strategies early in an epidemic or pandemic. (Feldmann et al., mBio, under review) (3) To identify and characterize host responses to viral infection to develop therapeutics: We have tested efficacy of three monoclonal antibodies (mAb) as a treatment for MERS-CoV infection in the common marmoset MERS disease model. Previously, these mAb had shown efficacy in mouse models of MERS-CoV infection. Unfortunately, none of the mABs showed significant reduction in disease burden and viral lung load in the nonhuman primate model suggesting that treatment with mABs may likely not very efficacious. However, there is a benefit of these mAbs for prophylactic applications. (de Wit et al. Antiviral Res 2018; de Wit et al., Antiviral Res 2019) We screened and characterized monoclonal antibodies (mAbs; human- and mouse-derived) for their capacity to bind and neutralize ANDV in vitro. We examined the most promising mAbs for post-exposure efficacy as a mono- or combined therapy in the Syrian hamster model of ANDV-induced Hantavirus Cardiopulmonary Syndrome (HCPS). Several mAbs completely protected hamsters from a lethal challenge. These data suggest that mono- or a cocktail-therapy could be effective post-exposure treatment for patients infected with ANDV-induced HCPS. (Garrido et al., Sc Transl Med 2018; Duehr et al. Nat Microbiol, submitted; Williamson et al., unpublished). (4) To develop protective vaccines: For MERS, we continued testing a promising DNA vaccine platform encoding a codon-optimized consensus spike protein. This vaccine induced potent cellular immunity and antigen specific neutralizing antibodies in three animal species using a prime/boost/boost approach. Vaccinated macaques were protected against MERS-CoV challenge and did not show any clinical or radiographic signs of pneumonia. Recently, we were successful in shortening the vaccination strategy for potential application of this vaccination approach in emergency situations to prevent MERS-CoV infection. The vaccine is now funded through the Coalition for Epidemic Preparedness Innovations (CEPI) under the lead of Inovio Pharmaceuticals. Further refinement of vaccine administration has been performed. (Falzarano et al, manuscript in preparation; de Wit et al., unpublished) To generate a vaccine against Nipah virus infection, we used the VSV platform to express single Nipah virus glycoproteins (G or F) as the immunogens. The vaccines elicited strong antibody responses in hamsters and nonhuman primates and protected them from lethal challenge. Since all recent outbreaks in humans were caused by the Bangladesh genotype, we developed new vectors that express the Nipah virus Bangladesh F or G. Those new vectors provided complete protection against disease in hamsters and nonhuman primates. In nonhuman primates, protection was achieved against homologous and heterologous challenge. The vaccine candidate expressing the Nipah virus G (Bangladesh) is now funded through CEPI under the lead of Public Health Vaccine (PHV). In future, we plan further evaluation of the vaccine in the African green monkey model (de Wit et al., manuscript in preparation) We generated several recombinant vesicular stomatitis virus (rVSV)-based H5N1 influenza vaccine vectors to demonstrate the feasibility of this platform for a fast-acting pan-H5 influenza virus vaccine. We chose multiple approaches regarding antigen design and genome location to define a more optimized vaccine approach. We found that a single dose of rVSV vectors expressing full-length hemagglutinin (HAfl) were enough to provide 100% protection against lethal H5N1 challenge. The vaccine vectors were fast-acting as demonstrated by uniform protection when administered 3 days prior to lethal challenge. Moreover, the vaccine vectors induced H5-specific antibodies with cross-hemagglutination inhibition for various H5-clade viruses, highlighting the potential of the VSV-based HAfl as a pan-H5 influenza virus emergency vaccine. Effective vaccines against H5N1 viruses are needed to counter the potential threat of a global pandemic caused by these pathogens. (Asada et al., NPJ Vaccines, in revision)

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de Wit, Emmie; Feldmann, Friederike; Okumura, Atsushi et al. (2018) Prophylactic and therapeutic efficacy of mAb treatment against MERS-CoV in common marmosets. Antiviral Res 156:64-71
Malachowa, Natalia; Freedman, Brett; Sturdevant, Daniel E et al. (2018) Differential Ability of Pandemic and Seasonal H1N1 Influenza A Viruses To Alter the Function of Human Neutrophils. mSphere 3:
de Wit, Emmie; Siegers, Jurre Y; Cronin, Jacqueline M et al. (2018) 1918 H1N1 Influenza Virus Replicates and Induces Proinflammatory Cytokine Responses in Extrarespiratory Tissues of Ferrets. J Infect Dis 217:1237-1246
Prescott, Joseph; Falzarano, Darryl; de Wit, Emmie et al. (2018) Pathogenicity and Viral Shedding of MERS-CoV in Immunocompromised Rhesus Macaques. Front Immunol 9:205
Falzarano, Darryl; Kamissoko, Badian; de Wit, Emmie et al. (2017) Dromedary camels in northern Mali have high seropositivity to MERS-CoV. One Health 3:41-43
de Wit, Emmie; Feldmann, Friederike; Horne, Eva et al. (2017) Domestic Pig Unlikely Reservoir for MERS-CoV. Emerg Infect Dis 23:985-988
van Doremalen, Neeltje; Falzarano, Darryl; Ying, Tianlei et al. (2017) Efficacy of antibody-based therapies against Middle East respiratory syndrome coronavirus (MERS-CoV) in common marmosets. Antiviral Res 143:30-37
de Wilde, Adriaan H; Falzarano, Darryl; Zevenhoven-Dobbe, Jessika C et al. (2017) Alisporivir inhibits MERS- and SARS-coronavirus replication in cell culture, but not SARS-coronavirus infection in a mouse model. Virus Res 228:7-13
Prescott, Joseph; Feldmann, Heinz; Safronetz, David (2017) Amending Koch's postulates for viral disease: When ""growth in pure culture"" leads to a loss of virulence. Antiviral Res 137:1-5
Borisevich, Viktoriya; Lee, Benhur; Hickey, Andrew et al. (2016) Escape From Monoclonal Antibody Neutralization Affects Henipavirus Fitness In Vitro and In Vivo. J Infect Dis 213:448-55

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